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ABSTRACT Electroactive organisms contribute to metal cycling, pollutant removal, and other redox-driven environmental processes via extracellular electron transfer (EET). Unfortunately, developing genotype-phenotype relationships for electroactive organisms is challenging because EET is necessarily removed from the cell of origin. Microdroplet emulsions, which encapsulate individual cells in aqueous droplets, have been used to study a variety of extracellular phenotypes but have not been applied to investigate EET. Here, we describe the development of a microdroplet emulsion system to sort and enrich EET-capable organisms from complex populations. We validated our system using the model electrogenShewanella oneidensisand described the tooling of a benchtop microfluidic system for oxygen-limited conditions. We demonstrated the enrichment of strains exhibiting electroactive phenotypes from mixed wild-type and EET-deficient populations. As a proof-of-concept application, we collected samples from iron sedimentation in Town Lake (Austin, TX) and subjected them to microdroplet enrichment. We measured an increase in electroactive organisms in the sorted population that was distinct compared to a population growing in bulk culture with Fe(III) as the sole electron acceptor. Finally, two bacterial species not previously shown to be EET-capable,Cronobacter sakazakiiandVagococcus fessus, were further cultured and characterized for electroactivity. Our results demonstrate the utility of microdroplet emulsions for isolating and identifying EET-capable bacteria.IMPORTANCEThis work outlines a new high-throughput method for identifying electroactive bacteria from mixed populations. Electroactive bacteria play key roles in iron trafficking, soil remediation, and pollutant degradation. Many existing methods for identifying electroactive bacteria are coupled to microbial growth and fitness—as a result, the contributions from weak or poor-growing electrogens are often muted. However, extracellular electron transfer (EET) has historically been difficult to study in high-throughput in a mixed population since extracellular reduction is challenging to trace back to the parent cell and there are no suitable fluorescent readouts for EET. Our method circumvents these challenges by utilizing an aqueous microdroplet emulsion wherein a single cell is statistically isolated in a pico- to nano-liter-sized droplet. Then, via fluorescence obtained from copper reduction, the mixed population can be fluorescently sorted and gated by performance. Utilizing our technique, we characterize two previously unrecognized weak electrogensVagococcus fessusandCronobacter sakazakii. 

ABSTRACT Advanced genome editing technologies have enabled rapid and flexible rewriting of theEscherichia coligenome, benefiting fundamental biology and biomanufacturing. Unfortunately, some of the most useful technologies to advance genome editing inE. colihave not yet been ported into other bacterial species. For instance, the addition of bacterial retrons to the genome editing toolbox has increased the efficiency of recombineering inE. coliby enabling sustained, abundant production of ssDNA recombineering donors by reverse transcription that install flexible, precise edits in the prokaryotic chromosome. To extend the utility of this technology beyondE. coli, we surveyed the portability and versatility of retron-mediated recombineering across three different bacterial phyla (Proteobacteria, BacillotaandActinomycetota) and a total of 15 different species. We found that retron recombineering is functional in all species tested, reaching editing efficiencies above 20% in six of them, above 40% in three of them, and above 90% in two of them. We also tested the extension of the recombitron architecture optimizations and strain backgrounds in a subset of hosts to additionally increase editing rates. The broad recombitron survey carried out in this study forms the basis for widespread use of retron-derived technologies through the whole Bacteria domain. 

We present Jammed Interconnected Bilayer Emulsions (JIBEs) as a class of tissue-like materials with macroscopic scalability and rapid fabrication, comprising millions to billions of bilayer-separated aqueous compartments. These materials closely mimic the organizational structure and properties of biological tissues. Our rapid self-assembly method for producing JIBEs generates milliliter- to deciliter-scale volumes within minutes representing over 10,000-fold improvement in the fabrication speed of droplet-based artificial tissues compared to existing droplet-based methods, enabling the creation of a truly macroscopic material. The method is highly adaptable to a wide range of amphiphiles, including lipids and block-copolymers, providing flexibility in tailoring JIBEs for diverse applications. The jammed architecture of JIBEs imparts unique properties, such as direct 3D-printabilty into aqueous solutions or onto air-exposed surfaces. Their membrane-bound structure also allows functionalization with biological and artificial nanochannels, enabling the material to exhibit the specific properties of the incorporated channels. In this work, we demonstrate three key features of JIBEs using distinct ion channels: tunable conductance, selective transport, and memristance. Incorporating an E. coli outer membrane protein increased ionic conductance by approximately 4,400-fold compared to non-functionalized tissues. Introducing a peptide-based transporter produced ion-selective membranes capable of discriminating ammonium over sodium at a ratio greater than 15:1. Finally, incorporating a model voltage-gated pore enabled the construction of a massively networked memristive device. We propose that functionalizing JIBEs with additional membrane proteins or synthetic ion channels could unlock a broad range of applications, including separations, energy generation and storage, neuromorphic computing, tissue engineering, drug delivery, and soft robotics. 

Engineered living materials combine the advantages of biological and synthetic systems by leveraging genetic and metabolic programming to control material-wide properties. Here, we demonstrate that extracellular electron transfer (EET), a microbial respiration process, can serve as a tunable bridge between live cell metabolism and synthetic material properties. In this system, EET flux from Shewanella oneidensis to a copper catalyst controls hydrogel cross-linking via two distinct chemistries to form living synthetic polymer networks. We first demonstrate that synthetic biology-inspired design rules derived from fluorescence parameterization can be applied toward EET-based regulation of polymer network mechanics. We then program transcriptional Boolean logic gates to govern EET gene expression, which enables design of computational polymer networks that mechanically respond to combinations of molecular inputs. Finally, we control fibroblast morphology using EET as a bridge for programmed material properties. Our results demonstrate how rational genetic circuit design can emulate physiological behavior in engineered living materials. 

Abstract Organic electrochemical transistors (OECTs) are ideal devices for translating biological signals into electrical readouts and have applications in bioelectronics, biosensing, and neuromorphic computing. Despite their potential, developing programmable and modular methods for living systems to interface with OECTs has proven challenging. Here we describe hybrid OECTs containing the model electroactive bacteriumShewanella oneidensisthat enable the transduction of biological computations to electrical responses. Specifically, we fabricated planar p-type OECTs and demonstrated that channel de-doping is driven by extracellular electron transfer (EET) fromS. oneidensis. Leveraging this mechanistic understanding and our ability to control EET flux via transcriptional regulation, we used plasmid-based Boolean logic gates to translate biological computation into current changes within the OECT. Finally, we demonstrated EET-driven changes to OECT synaptic plasticity. This work enables fundamental EET studies and OECT-based biosensing and biocomputing systems with genetically controllable and modular design elements.